y Heterogeneous translational landscape of the endoplasmic reticulum revealed by ribosome proximity labeling and transcriptome analysis
y Heterogeneous translational landscape of the endoplasmic reticulum revealed by ribosome proximity labeling and transcriptome analysis
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DOI：
10.1074/jbc.ra119.007996
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发表时间：
2019-05-31
影响因子：
4.8
通讯作者：
Nicchitta, Christopher V.
中科院分区：
文献类型：
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作者：
Hoffman, Alyson M.;Chen, Qiang;Nicchitta, Christopher V.
内质网（ER）是mRNA定位和翻译的纽带，最近的研究表明，ER结合的核糖体在调节蛋白质组组成中也起着转录组范围的作用。Sec 61易位子（SEC 61）作为核糖体的受体，其翻译分泌/整合膜蛋白编码mRNA，但SEC 61是否也作为胞质蛋白编码mRNA的翻译位点仍然未知。在这里，使用BioID邻近标记方法在HEK 293 T Flp-In细胞系中，我们研究了ER驻留蛋白和核糖体在体内的相互作用。使用体外分析，我们进一步集中在真正的核糖体相互作用（即SEC 61）和ER蛋白（核糖体蛋白I，富含亮氨酸的重复序列59（LRRC 59），和SEC 62）先前涉及与核糖体。我们观察到ER结合的核糖体的标记与SEC 61和LRRC 59 BioID报告，比较温和的标记与ribophorin I报告，和没有标记与SEC 62报告。一种生物素脉冲追踪/亚细胞分级分离方法，检查核糖体交换在SEC 61和LRRC 59网站显示，在稳态下，核糖体在这些网站包括快速和缓慢交换池。由抑制剂三尖杉酯碱引起的全局翻译起始停滞加速了SEC 61标记的核糖体交换。与SEC 61和LRRC 59标记的核糖体相关的mRNA的RNA-Seq分析揭示了位点富集和共享的mRNA，并进一步确定了ER在调节蛋白质组组成中具有转录组范围的作用。这些结果提供了证据，核糖体通过多种模式与ER膜相互作用，并建议通过ER膜结合核糖体控制全球蛋白质组组成的调节机制。
The endoplasmic reticulum (ER) is a nexus for mRNA localization and translation, and recent studies have demonstrated that ER-bound ribosomes also play a transcriptome-wide role in regulating proteome composition. The Sec61 translocon (SEC61) serves as the receptor for ribosomes that translate secretory/integral membrane protein-encoding mRNAs, but whether SEC61 also serves as a translation site for cytosolic protein-encoding mRNAs remains unknown. Here, using a BioID proximity-labeling approach in HEK293T Flp-In cell lines, we examined interactions between ER-resident proteins and ribosomes in vivo. Using in vitro analyses, we further focused on bona fide ribosome interactors (i.e. SEC61) and ER proteins (ribophorin I, leucine-rich repeat-containing 59 (LRRC59), and SEC62) previously implicated in associating with ribosomes. We observed labeling of ER-bound ribosomes with the SEC61 and LRRC59 BioID reporters, comparatively modest labeling with the ribophorin I reporter, and no labeling with the SEC62 reporter. A biotin pulse-chase/subcellular fractionation approach to examine ribosome exchange at the SEC61 and LRRC59 sites revealed that, at steady state, ribosomes at these sites comprise both rapid- and slow-exchanging pools. Global translational initiation arrest elicited by the inhibitor harringtonine accelerated SEC61 reporter-labeled ribosome exchange. RNA-Seq analyses of the mRNAs associated with SEC61- and LRRC59-labeled ribosomes revealed both site-enriched and shared mRNAs and further established that the ER has a transcriptome-wide role in regulating proteome composition. These results provide evidence that ribosomes interact with the ER membrane via multiple modes and suggest regulatory mechanisms that control global proteome composition via ER membrane-bound ribosomes.